Abstract

Fermentative and respiratory yeast strains of genera Saccharomyces, Kluyveromyces, Pichia, Candida and Hansenula have been investigated for mitochondrial localization of Cu/Zn superoxide dismutase (SOD). Pure mitochondrial fractions were obtained and the specific activities of Cu/Zn and Mn SODs were measured in comparison with those in the corresponding cell-free extracts. The Cu/Zn SOD: Mn SOD ratio in mitochondria and crude extracts was calculated and was considered a specific characteristic of all tested strains. Electrophoretical visualization of SOD patterns provided evidence for possible migration of cytosolic Cu/Zn SOD to mitochondria. The characteristic Cu/Zn SOD profile in mitochondria of all tested strains suggested its ubiquity within the fermentative and respiratory yeasts.

Keywords

Fermentative/respiratory yeast

Mitochondrion

Superoxide dismutase

1 Introduction

Copper- and zinc-containing superoxide dismutases (Cu/Zn SODs) are characteristic for cytosol of eukaryotic cells [1]. Weisinger and Fridovich described the presence of these enzymes in intermembrane space of mitochondria isolated from the yeast Saccharomyces cerevisiae[2]. Recently, Sturtz et al., continuing investigation of the S. cerevisiae model system, have studied the localization and function of Cu/Zn SOD enzyme in mitochondria [3]. They have found that the protein located in this compartment did not contain typical N-terminal presequences for mitochondrial uptake, but its accumulation in this organelle is strongly influenced by the corresponding cooper chaperone. Using sod1Δ mutant they demonstrated that Cu/Zn SOD localized in mitochondria protects proteins from oxidative injury, as the mutant showed increased carbonylation damage when compared with the wild-type strain.

The essential role of mitochondrial Cu/Zn SOD in S. cerevisiae yeasts put forward the question about its ubiquity within other types of yeasts, as they exhibit divergences in their modes of oxygen uptake and energy generation [4]. There are several groups of yeasts in respect to their utilization of respiration and fermentation in ATP production. Considering the destiny of pyruvate as a product of glucose catabolism yeasts can be divided into two groups: obligate aerobes and facultative anaerobes [5]. The group of facultative anaerobes consists of two subgroups: fermentative and respiratory yeasts. The distinction between these subgroups applies mainly to aerobic growth on glucose, fructose and mannose. Its molecular basis is related to repression of several respiratory enzymes by the sugars in the fermentative subgroup [5]. It is important to know whether, besides S. cerevisiae, other strains from this subgroup and those belonging to respiratory ones also possess Cu/Zn enzyme, localized in mitochondria.

In the present study we have investigated the mitochondrial localization of Cu/Zn SOD using yeasts of different genera: Saccharomyces, Kluyveromyces, Pichia, Candida and Hansenula belonging to fermentative and respiratory subgroups. We demonstrate here that pure mitochondrial fraction, obtained from representative strains of these genera, possesses Cu/Zn SOD enzymes, and evidence is provided for its ubiquity within the fermentative and respiratory types of yeasts.

Cells were harvested by centrifugation at 800×g for 10 min and washed twice with distilled H2O. Cell wall disruption was carried out by spheroplasting according to the procedure of Defontaine et al. [6]. The cell debris was removed by centrifugation at 1000×g for 10 min and the cell-free extracts were triply frozen and thawed for mitochondria disruption, and centrifuged at 15 000×g for 10 min. The supernatants obtained were used for enzymatic analyses.

The fraction of heavy mitochondria was isolated from the spheroplasts after osmotic shock in distilled water applying the procedure of Holtta et al. [7]. The cell debris was harvested at 1000×g for 10 min and the heavy mitochondrial fraction was isolated by centrifugation at 3500×g for 20 min. Then the crude mitochondrial pellet was carefully washed with buffer, containing 0.5 M sorbitol, 50 mM Tris, 10 mM EDTA, pH 7.5. This procedure was repeated three times and the collected mitochondria were resuspended in deionized water and lysed by freezing and thawing. Thus obtained sample of disrupted mitochondria was subjected to further biochemical analyses.

2.3 Biochemical analyses

SOD [EC: 1.15.1.1] activity was measured as described by Beauchamp and Fridovich [8]. One unit was defined as the amount of enzyme causing 50% decrease in the reduction of nitro blue tetrazolium (NBT) to blue formazan under the test conditions. Cu/Zn and Mn type of SOD enzymes were distinguished by inhibition with 2 and 5 mM KCN [9]. In order to detect an interference of Fe-containing SOD a treatment with 5 mM H2O2 was performed according to the procedure of Britton et al. [10].

Succinate dehydrogenase [1.3.99.1] activity was determined by the method of Bonner [11]. One unit of enzyme activity was expressed as mmol reduced K3Fe(CN)6 min−1 mg protein−1.

d-Amino acid oxidase [1.4.3.3] activity was measured, as described by Lichtenberg and Wellner [13]. One-tenth unit of activity yields an absorbancy of 0.180 at λ=300 nm.

Isocitric lyase [4.1.3.1] activity was assayed according to the procedure of Daron and Gunsalus [14]. One unit was defined as the amount of enzyme catalyzing the formation of 1 μmol glyoxylate for 5 min at 28°C, at pH 6.0.

The standard deviation in enzyme activities was estimated at least in three independent experiments and calculated according to the criterion of Student.

2.4 Protein determination

Protein content was determined by the method of Lowry et al. [15]. Bovine serum albumin (Sigma St. Louis, MO, USA) was used as a standard.

2.5 Polyacrylamide gel electrophoresis (PAGE) and immunoblotting

PAGE and specific staining for SOD including inhibition analysis were performed as described previously [16]. The mitochondrial fraction was concentrated with the Centricon (Amicon) device with a membrane cut-off of 10 kDa. Sodium dodecyl sulfate–PAGE and immunoblotting were performed according to Riffer et al., [17]. The following primary antibodies against marker proteins of S. cerevisiae were obtained from Molecular Probes (Leiden, The Netherlands): mouse monoclonal anti-Cox3p (cytochrome c oxidase subunit III), mouse monoclonal Pep12p (endosomal membrane protein) and mouse anti-PGK (polyglycerate kinase). Alkaline phosphatase-conjugated anti-mouse IgG was used as secondary antibody (Sigma-Aldrich, Germany) and colorimetric signal detection of the immunoprecipitate was performed using a NBT/BCIP stock solution (Roche, Mannheim, Germany) diluted 1:500 in staining buffer.

3 Results and discussion

3.1 Activity of Cu/Zn- and Mn-containing SOD enzymes in crude extracts and mitochondria of fermentative and respiratory yeasts

The investigation has been performed using two groups of yeasts: fermentative (S. cerevisiae and S. uvarum), which are able to grow aerobically on glucose with very a high catabolic rate (100–300 mmol glucose g−1 min−1) and a low rate of oxygen consumption (5–50 mmol O2 g−1 min−1), and respiratory, represented by typical examples of genera Hansenula, Pichia, Kluyveromyces and Candida. They differ from the fermentative ones possessing a very high respiration rate during utilization of glucose (150–200 mmol O2 g−1 min−1) and a very low rate of glucose catabolism (10–40 mmol glucose g−1 min−1) [5].

Crude mitochondrial fractions from all these model strains have been obtained and tested for purity through measurement of the specific activities of several enzymes, typically located in different cellular compartments: hexokinase and glucose-6-phosphate dehydrogenase in cytosol, d-amino acid oxidase and isocitric lyase in peroxisomes and succinate dehydrogenase and Mn SOD in mitochondria. Results are presented in Table 1. Using these enzymes, the purity of mitochondrial fractions was displayed, as enzyme activities, characteristic for the cytosol and the peroxisomes, were not detected there. The measured values for the activity of succinate dehydrogenase and Mn SOD were typical for a mitochondrial fraction [18], which proved that it is not contaminated with enzyme activities characteristic for other cellular compartments. This has been additionally proved using Western blot analysis. An aliquot of S. cerevisiae NBIMCC 583 crude mitochondrial fraction has been probed with monoclonal antibodies against marker mitochondrial, endosomal and cytosolic proteins as follows: cytochrome c oxidase for mitochondria, a membrane protein for endosomes and polyglycerate kinase for the cytosol. The data are presented in Fig. 1. It can be seen that the obtained pattern confirmed the purity of mitochondrial fractions proved by the enzymatic tests.

The total SOD activity was measured both in mitochondrial fractions and cell-free extracts. The data indicated in Table 1 show that it was higher in the respiratory type of yeasts correlating with the mode of glucose metabolism [5]. The part of the specific Cu/Zn SOD enzyme in the obtained crude extracts and mitochondrial fractions was determined applying inhibition assay. The data are presented in Table 2. These results showed that Cu/Zn SOD activity in mitochondria of the investigated strains was between 4.5 and 6.5 U mg protein−1 and the one measured in the cell-free extracts was between 12.5 and 22.5 U mg protein−1. At the same time the Mn SOD enzyme ranges from 14.5 to 26.0 U mg protein−1 in the mitochondrial fractions and from 3.5 to 10.5 U mg protein−1 in the crude extracts. A comparison of the relative part (in %) of the Cu/Zn SOD and Mn SOD enzymes was performed both in cell-free extracts and mitochondrial fractions of all tested strains. The data obtained are presented in Table 3. It can be seen that the percentage of the Cu/Zn SOD in mitochondria varies between 15–29%, while that in the crude extract is around 70–85%, which correlates with other investigations [3]. On the basis of the distribution of Cu/Zn and Mn SOD activities (in %) an intriguing ratio between the two form of the enzyme was determined. Summarizing the data from Table 3 it could be concluded that this ratio possesses several characteristics: (1) it is a reciprocal one regarding the values estimated for the cell-free extracts and mitochondria (ranging between 2.0 and 3.5 and between 0.2 and 0.4, respectively); (2) its value is higher for the fermentative strains S. cerevisiae and S. uvarum. It seems that Cu/Zn and Mn SOD ratio in the cell-free extracts and mitochondria is a specific characteristic of all studied strains, indicating that both enzymes possessed a concerted action in yeast cells and particularly in their mitochondria.

The total SOD activity in the corresponding sample is accepted as 100%.

3.2 Visualization of mitochondrial SOD

The mitochondrial fractions and crude extracts obtained from representatives of both fermentative and respiratory strains were subjected to 10% PAGE. The results obtained are presented in Fig. 2. All mitochondrial fractions represent specifically stained for SOD bands. After treatment with 2 and 5 mM KCN the Cu/Zn SOD is inhibited. An additional processing of the samples with 5 mM H2O2 for discrimination of Fe SOD (Fig. 3) indicated that the already obtained profile for SOD enzymes described in Fig. 2 is not changed. Evidently, a band in crude extracts is relevant to a cyanide-sensitive one with the same Rm value, visible in the mitochondrial fraction. In this context it is reasonable to conclude that in all investigated yeast strains the Cu/Zn SOD enzyme, characteristic for cytosol, migrates to mitochondria. This probably happens through the mechanisms described by Sturtz et al. [3] for mitochondrial localization of Cu/Zn SOD enzyme in S. cerevisiae.

The characteristic profile of Cu/Zn enzymes in mitochondria of all tested strains suggests that the presence of the isoenzyme in these organelles is a common phenomenon for yeasts, regardless of the type of their energy yielding metabolism. The existence of Cu/Zn SOD in yeast mitochondria supports the findings of Okado-Matsumoto and Fridovich [19] for presence of this enzyme in mitochondrial intermembrane space of higher eukaryotic cells (rat liver), coded by the same gene as the cytosolic Cu/Zn SOD.

All these data make possible a speculation about the general role of Cu/Zn SOD in regulation and processing of enzymes responsible for antioxidant defense of the eukaryotic cells.